Stimulation of cartilage growth with agonists of the non-proteolytically activated thrombin receptor

ABSTRACT

Disclosed is a method of stimulating cartilage growth, repair or regeneration at a site in a subject in need of such growth, repair or regeneration. The method comprises the step of administering a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor to the site. 
     Also disclosed is a method of stimulating the proliferation and expansion of chrondrocytes in vitro. The method comprises culturing chrondrocytes in the presence of a stimulating amount of an NPAR agonist.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/909,348 filed Jul. 19, 2001, which claims the benefit of U.S.Provisional Application No. 60/219,800. filed Jul. 20, 2000, the entireteachings of which are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant 1 R43AR46343-01 from the National Institutes of Health/National Institute ofArthritis and Muscoskeletal and Skin Diseases. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Unlike most tissues, cartilage does not self-repair following injury.Cartilage is an avascular tissue made up largely of cartilage specificcells, the chondrocytes, special types of collagen, and proteoglycans.The inability of cartilage to self-repair after injury, disease, orsurgery is a major limiting factor in rehabilitation of degrading jointsurfaces and injury to meniscal cartilage. Osteoarthritis, the majordegenerative disease of weight bearing joint surfaces, is caused byeroding or damaged cartilage surfaces and is present in approximately25% of the over 50-year-old population. In the US more than 20 millionpeople suffer from osteoarthritis, with annual healthcare costs of morethan $8.6 billion. In addition, the cost for cartilage repair from acutejoint injury (meniscal lesions, patellar surface damage andchondromalacia) exceeds $1 billion annually. Therefore, new therapeuticapproaches are needed to heal lesions of cartilage caused bydegeneration or acute trauma.

SUMMARY OF THE INVENTION

It has now been found that chondrocytes isolated from articularcartilage respond to compounds which activate the non-proteolyticthrombin cell surface receptor (hereinafter “NPAR”). For example,chondrocytes express approximately 233,000 thrombin binding sites percell with apparent affinities of approximately 0.1 nM (3000 sites) and27 nM (230,000 sites) (Example 1). In addition, the compound TP508, anagonist of the non-proteolytic thrombin receptor, stimulatesproliferation of bovine chondrocytes in culture in the presence ofthrombin as a co-mitogen (Example 2A) and stimulates by itself theproliferation of rat chondrocytes cultured in three dimensional matrixculture (Example 3A). This same TP508 compound also stimulatesproteoglycan synthesis as measured by the incorporation of ³⁵S sulfatein both bovine chondrocytes (Example 2B) and 3-dimensional cultures ofrat chondrocytes (Example 3B). These in vitro experiments demonstratethat NPAR agonists can stimulate proliferation and matrix production inchondrocytes isolated from articular cartilage. Additional in vivoexperiments demonstrate that delivering TP508 in a sustained releaseformulation to rabbit trochlear grove cartilage defects which extendinto the subchondral bone results in repair of the cartilage defect,including repair of subchondral bone, restoration of a normal cartilagesurface and integration of the newly formed cartilage with uninjuredcartilage outside of the defect area (Example 5).

Based on the results reported in the prior paragraph, novel methods ofstimulating chondrocyte growth in vivo and cartilage repair in a subjectand novel delivery methods for delivering pharmaceutical compositions toarticular defects to aid in surface repair and to prevent articulardegradation are disclosed herein.

The present invention is a method of stimulating cartilage growth,regeneration or repair at a site in a subject where cartilage growth,repair or regeneration is needed. The method comprises the step ofadministering a therapeutically effective amount of an agonist of thenon-proteolytically activated thrombin receptor to the site of injury.

Another embodiment of the present invention is a method of stimulatingthe proliferation and expansion of chrondrocytes in vitro. The methodcomprises culturing chrondrocytes in the presence of a stimulatingamount of an NPAR agonist.

DETAILED DESCRIPTION OF THE INVENTION

Sites in need of cartilage growth, repair or regeneration are found insubjects with osteoarthritis. Osteoarthritis or degenerative jointdisease is a slowly progressive, irreversible, often monoarticulardisease characterized by pain and loss of function. The underlying causeof the pain and debilitation is the cartilage degradation that is one ofthe major symptoms of the disease. Hyaline cartilage is a flexibletissue that covers the ends of bones and lies between joints such as theknee. It is also found in between the bones along the spine. Cartilageis smooth, allowing stable, flexible movement with minimal friction, butis also resistant to compression and able to distribute applied loads.As osteoarthritis progresses, surfaces of cartilage and exposedunderlying bone become irregular. Instead of gliding smoothly, boneyjoint surfaces rub against each other, resulting in stiffness and pain.Regeneration of damaged cartilage and the growth of new cartilage atthese arthritic sites would relieve the pain and restore the loss offunction associated with osteoarthritis.

Cartilage damage can also occur from trauma resulting from injury orsurgery. Sports injuries are a common cause of cartilage damage,particularly to joints such as the knee. Traumatic injury to cartilagecan result in the same type of functional impairment. Therefore, sitesin a subject with cartilage that has been damaged by trauma or diseaseare in need of treatment to restore or promote the growth of cartilage.

Applicants have discovered that compounds which stimulate or activatethe non-proteolytically activated thrombin receptor (hereinafter “NPAR”)can stimulate chondrocytes to proliferate. Chondrocytes are cells whichmake up about 1% of the volume of cartilage and which replace degradedmatrix molecules to maintain the correct volume and mechanicalproperties of the tissue. Applicants have also found that compoundswhich stimulate or activate NPAR stimulate proteoglycan synthesis inchondrocytes. Proteoglycan is a major cartilage component. Based onthese results, Applicants delivered the NPAR agonist TP508, prepared ina sustained release formulation, to defects in rabbit trochlear grovecartilage and discovered that the peptide stimulated repair of thedefect that included formation of new cartilage with a normal cartilagesurface. The peptide also stimulated layering and integration of thisnew cartilage into adjacent, uninjured cartilage and restoration of thesubchondral bone. It is concluded that NPAR agonists can inducecartilage growth and repair when administered to sites needing cartilagegrowth and/or repair.

Compounds which stimulate or activate NPAR are said to be NPAR agonists.NPAR is a high-affinity thrombin receptor present on the surface of mostcells. NPAR is largely responsible for high-affinity binding ofthrombin, proteolytically inactivated thrombin, and thrombin derivedpeptides to cells. NPAR agonists and antagonists can compete for theaffinity binding with thrombin to cells (see, e.g., Glenn et al., J.Peptide Research 1:65 (1988)). NPAR appears to mediate a number ofcellular signals that are initiated by thrombin independent of itsproteolytic activity. An example of one such signal is the upregulationof annexin V and other molecules identified by subtractive hybridization(see Sower, et. al., Experimental Cell Research 247:422 (1999)). NPAR istherefore characterized by its high affinity interaction with thrombinat cell surfaces and its activation by proteolytically inactivederivatives of thrombin and thrombin derived peptide agonists asdescribed below. NPAR activation can be assayed based on the ability ofits agonists to stimulate cell proliferation when added to fibroblastsin the presence of submitogenic concentrations of thrombin or moleculesthat activate protein kinase C as disclosed in U.S. Pat. Nos. 5,352,664and 5,500,412.

NPAR is to be distinguished from other thrombin binding proteins and thecloned family of proteolytically-activated receptors for thrombin,including the receptors PAR1, PAR2, PAR3 and PAR4. PAR1 possesses aspecific thrombin cleavage site that allows thrombin cleavage to exposea new amino-terminus domain that acts as a tethered ligand folding backonto itself inducing its activation (see, Vu, et al., Cell. 64:1057(1991)). PAR2 has a similar mechanism for activation, but is principallyactivated by trypsin-like enzymes (see, Zhong, et al., J. Biol. Chem.267:16975 (1992)). PAR3 also has a similar mechanism of activation andappears to function as a second thrombin receptor in platelets (see,Ishihara, et al., Nature. 386:502 (1997)). PAR4 has been detected inmouse megakaryocytes and studies suggest that it also functions in humanplatelets (see, Kahn, et al., Nature 394:690 (1998)). In contrast withthese PAR receptors, activation of NPAR requires no proteolyticcleavage.

Several lines of evidence indicate that NPAR is distinct from PARreceptors: (1) a population of cells has been isolated that expressfully functional PAR1 receptors, but are non-responsive to thrombin dueto a defect in the NPAR signal transduction pathway (see, Kim, et al.,J. Cell. Physiol. 160:573 (1994)); (2) neutrophils bind ¹²⁵I thrombinwith high affinity and their chemotaxis is stimulated by proteolyticallyinactivated thrombin or NPAR agonists (see, Ramakrishnan and Carney,Mol. Biol. Cell 4:1993 (1993)), yet they do not express PAR1 (seeJenkins, et al., J. Cell Sci. 108:3059 (1995)); (3) IIC9 fibroblastsover-express PAR1, but do not bind thrombin with high affinity (see,Kim, D. Ph.D. Dissertation. The University of Texas Medical Branch atGalveston, 1995; and Low, et al., “Cancer Cells 3/Growth Factors andTransformation”, Cold Spring Harbor Laboratory, New York); and (4) NPARagonists have distinct effects on gene expression from those of the PARreceptor agonist peptides (see, Sower, et. al., Experimental CellResearch 247: 422 (1999).

One example of an NPAR agonist is a thrombin peptide derivative andphysiologically functional equivalents. i.e., a polypeptide with no morethan about fifty amino acids, preferably no more than about thirty aminoacids and having sufficient homology to the fragment of human thrombincorresponding to prothrombin amino acids 508-530 (SEQ ID NO: 5) that thepolypeptide activates NPAR. The thrombin peptide derivatives describedherein preferably have between about 12 and 23 amino acids, morepreferably between about 19 and 23 amino acids. One example of athrombin peptide derivative comprises a moiety represented by Structuralformula (I):Asp-Ala-R  (I)R is a serine esterase conserved domain. Serine esterases, e.g.,tyrpsin, thrombin, chymotrypsin and the like, have a region that ishighly conserved. “Serine esterase conserved domain” refers to apolypeptide having the amino acid sequence of one of these conservedregions or is sufficiently homologous to one of these conserved regionssuch that the thrombin peptide derivative retains NPAR activatingability.

A physiologically functional equivalent of a thrombin derivativeencompasses molecules which differ from thrombin derivatives inparticulars which do not affect the function of the thrombin receptorbinding domain or the serine esterase conserved amino acid sequence.Such particulars may include, but are not limited to, conservative aminoacid substitutions and modifications, for example, amidation of thecarboxyl terminus, acetylation of the amino terminus, conjugation of thepolypeptide to a physiologically inert carrier molecule, or sequencealterations in accordance with the serine esterase conserved sequences.

A thrombin receptor binding domain is defined as a polypeptide whichdirectly binds to the thrombin receptor and/or competitively inhibitsbinding between high-affinity thrombin receptors and alpha thrombin.

In one embodiment, the serine esterase conserved sequence has the aminoacid sequence of SEQ ID NO: 1 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val)or a C-terminal truncated fragment of a polypeptide having the aminoacid sequence of SEQ ID NO: 1. It is understood, however, that zero,one, two or three amino acids in the serine esterase conserved sequencecan differ from the corresponding amino acid in SEQ ID-NO: 1.Preferably, the amino acids in the serine esterase conserved sequencewhich differ from the corresponding amino acid in SEQ ID NO: 1 areconservative substitutions, and are more preferably highly conservativesubstitutions. A “C-terminal truncated fragment” refers to a fragmentremaining after removing an amino acid or block of amino acids from theC-terminus, said fragment having at least six and more preferably atleast nine amino acids.

More preferably, the serine esterase conserved sequence has the aminoacid sequence of SEQ ID NO: 2 (Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val; X₁is Glu or Gln and X₂ is Phe, Met, Leu, His or Val) or a C-terminaltruncated fragment thereof having at least six amino acids, preferablyat least nine amino acids.

In a preferred embodiment, the thrombin peptide derivative comprises aserine esterase conserved sequence and a polypeptide having a morespecific thrombin amino acid sequence Arg-Gly-Asp-Ala (SEQ ID NO: 3).One example of a thrombin peptide derivative of this type comprisesArg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val (SEQ ID NO: 4). X₁and X₂ are as defined above. When the thrombin peptide derivativecomprises SEQ ID NO: 4, it preferably has the amino acid sequence of SEQID NO: 5(Ala-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val)or an N-terminal truncated fragment thereof, provided that zero, one,two or three amino acids at positions 1-9 in the thrombin peptidederivative differ from the amino acid at the corresponding position ofSEQ ID NO: 5. Preferably, the amino acids in the thrombin peptidederivative which differ from the corresponding amino acid in SEQ ID NO:5 are conservative substitutions, and are more preferably highlyconservative susbstitutions. An “N-terminal truncated fragment” refersto a fragment remaining after removing an amino acid or block of aminoacids from the N-terminus, preferably a block of no more than six aminoacids, more preferably a block of no more than three amino acids.

TP508 is an example of a thrombin peptide derivative and has the aminoacid sequence of SEQ ID NO: 5. A physiologically functional equivalentof SEQ ID NO: 5 is SEQ ID NO: 6 which has the identical amino acidsequence of SEQ ID NO: 5 and also contains a C-terminal amide.

A “conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in the their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Listed below are five groups of amino acids. Replacing an amino acid ina polypeptide with another amino acid from the same group results in aconservative substitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, and non-naturally occurring amino acids        with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic        side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, omithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C1-C4 aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

A “highly conservative substitution” is the replacement of an amino acidwith another amino acid that has the same functional group in the sidechain and nearly the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have nearly the same sizewhen the total number carbon and heteroatoms in their side chainsdiffers by no more than two. They have nearly the same shape when theyhave the same number of branches in the their side chains. Example ofhighly conservative substitutions include valine for leucine, threoninefor serine, aspartic acid for glutamic acid and phenylglycine forphenylalanine. Examples of substitutions which are not highlyconservative include alanine for valine, alanine for serine and asparticacid for serine.

Other NPAR agonists include small organic molecules which bind andactivate NPAR. Agonists of this type can be conveniently identified withhigh through-put screening, e.g., with assays that assess the ability ofmolecules to stimulate cell proliferation when added to fibroblasts inthe presence of submitogenic concentrations of thrombin or moleculesthat activate protein kinase C or with assays that assess the ability ofthese molecules to compete with ¹²⁵I-thrombin to cells with surface NPARreceptors, as disclosed in Glenn et al., supra, U.S. Pat. Nos. 5,352,664and 5,500,412. The entire teachings for Glenn et al., and U.S. Pat. Nos.5,352,664 and 5,500,412 are incorporated herein by reference.

The term “NPAR agonist” also includes compounds and combinations ofcompounds known to activate NPAR. Examples are disclosed in U.S. Pat.Nos. 5,352,664 and 5,500,412 and include thrombin andDIP-alpha-thrombin.

NPAR agonists used in the method of the present invention are typicallyadministered as one component in a pharmaceutical composition to thesite in need of cartilage growth, repair or regeneration. Administeringto the site in need of treatment means that the pharmaceuticalcomposition containing the NPAR agonist is administered in sufficientproximity to the site in need of treatment so that cartilage growth orcartilage regeneration occurs at the site (e.g., a greater amount ofcartilage growth or better quality of cartilage growth in the presenceof the NPAR agonist than in its absence).

In one means of administration, the pharmaceutical composition is asolution comprising the NPAR agonist and a suitable carrier. Thesolution is applied directly to or in near proximity to the site in needof treatment. Administration of the solution can be convenientlyaccomplished, for example, intraarticularly by syringe, in closeproximity to the damaged tissue by syringe or through a surgicalopening. Standard pharmaceutical formulation techniques may be employedsuch as those described in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. Suitable pharmaceutical carriers forinclude, for example, physiological saline, bacteriostatic saline(saline containing about 0.9% mg/ml benzyl alcohol), phosphate-bufferedsaline, Hank's solution, Ringer's-lactate and the like.

In another means of administration, the pharmaceutical compositioncomprises the NPAR agonist and an implantable biocompatible carrier. Abiocompatible carrier should be non-toxic, non-inflammatory,non-immunogenic and devoid of other undesired reactions at theimplantation site. Suitable carriers also provide for release of theactive ingredient and preferably for a slow, sustained release over timeat the implantation site.

A number of synthetic biodegradable polymers can serve as carriers withsustained release characteristics. Examples of these polymers includepoly α-hydroxy esters such as polylactic acid/polyglycolic acidcopolymers and polyanhydrides.

Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are wellknown in the art as sustained release vehicles. The rate of release canbe adjusted by the skilled artisan by variation of polylactic acid topolyglycolic acid ratio and the molecular weight of the polymer (seeAnderson, et al., Adv. Drug Deliv. Rev. 28:5 (1997), the entireteachings of which are incorporated herein by reference). Theincorporation of poly(ethylene glycol) into the polymer blend allowsfurther attenuation of the release profile of the active ingredient (seeCleek et al., J. Control Release 48:259 (1997), the entire teachings ofwhich are incorporated herein by reference). Suitable implantable PLGApolymers for use as carriers for cartilage growth factors are describedin U.S. Pat. Nos. 6,013,853, 5,607,474 and 5,876,452, the entireteachings of which are incorporated herein by reference.

Polyanhydrides, shown in Structural Formula (II), have well defineddegradation and release characteristics that can be controlled byincluding varying amounts of hydrophobic or hydrophilic monomers such assebacic acid and 1,3-bis(p-carboxyphenoxy)propane (see Leong et al., J.Biomed. Mater. Res. 19:941 (1985), the entire teachings of which areincorporated herein by reference). To improve mechanical strength,anhydrides are often copolymerized with imides to formpolyanhydride-co-imides. Examples of polyanhydride-co-imides that aresuitable for orthopaedic applications arepoly(trimellitylimido-glycine-co-1,6-bis(carboxyphenoxy)hexane andpyromellityimidoalanine: 1,6-bis(p-carboxyphenoxy)hexane copolymers.

The pharmaceutical compositions can be shaped as desired in anticipationof surgery or shaped by the physician or technician during surgery. Itis preferred to shape the matrix to span a tissue defect and to take thedesired form of the new tissue. In the case of cartilage repair of largedefects, it is desirable to use dimensions that span the defect. Afterimplantation, the material is slowly absorbed by the body and isreplaced by cartilage in the shape of or very nearly the shape of theimplant.

In one aspect, the carrier is a porous matrix into which progenitorcells may migrate. Cells can often attach to such porous matrices, whichcan then serve as a scaffolding for tissue growth and thereby acceleratethe rate of bone growth. Chondrocytes can be applied to such matricesprior to implant to further accelerate healing. Collagen or a collagengel is an example of a suitable porous matrix.

In another aspect, the carrier is a viscous solution or gel that isinjectable intraarticuarly or at the site in need of treatment.Hyaluronic acid is an example of a carrier of this type. Hyaluronic acidproducts are commercially available and include ORTHOVISC developed byAnika, SYNVISC, developed by Biomatrix, HYALGAN, developed by Fidia andARTZ, developed by Seikagaku. Pluronic gel is another example of thistype of carrier. Pluronic gels are nontxoic block copolymers of ethyleneoxide and propylene oxide. They exhibit thermosetting properties thatallow them to exist as viscous liquids at room temperatures, but as gelsat body temperatures. Injectable compositions can be applied directly tothe site in need of treatment without the need for invasive surgery.Polymers of poly(ethylene oxide) and copolymers of ethylene andpropylene oxide are also suitable as injectable matrices (see Cao et al,J. Biomater. Sci 9:475 (1998) and Sims et al., Plast Reconstr.Surg.98:843 (196), the entire teachings of which are incorporated herein byreference).

A “therapeutically effective amount” is the quantity of NPAR agonist (orchondrocytes) which results in greater cartilage growth or repair in thepresence of the NPAR agonist than in its absence. Alternatively oraddition, a “therapeutically effective amount” is the quantity of NPARagonist (or chondrocytes) which results in alleviation of the painand/or lack of function associated with the cartilage damage. Typically,the agonist (or chondrocytes) is administered for a sufficient period oftime to achieve the desired therapeutic or effect. The amountadministered will depend on the amount of cartilage growth that isdesired, the health, size, weight, age and sex of the subject and therelease characteristics of the pharmaceutical formulation. Typically,between about 0.1 μg per day and about 1 mg per day of NPAR agonist(preferably between about 5 μg per day and about 100 μg per day) isadministered by continuous release or by direct application to the sitein need of cartilage growth or repair.

A “subject” is preferably a human, but can also be an animal in need oftreatment, e.g., companion animals (e.g., dogs, cats, and the like),farm animals (e.g., cows, pigs, horses and the like) and laboratoryanimals (e.g., rats, mice, guinea pigs and the like).

NPAR agonists can be used to accelerate the growth or to maintain thefunctionality of isolated chondrocytes. In one embodiment, NPAR agonistscan be added to tissue culture medium to stimulate proliferation andprovide for more rapid proliferation and/or to prevent apoptotic deathor senescence of cells often encountered when primary cell isolates areplace in culture. In another embodiment, because the NPAR agonistsappear to stimulate matrix production, such NPAR agonists could be usedto maintain the differentiated functionality of chondrocytes in culture.NPAR agonists can be used alone in standard defined tissue culturemedium or as a supplement to tissue culture medium containing serum orother growth factor to provide additive or synergistic effects on the invitro production or maintenance of chondrocytes. A sufficient quantityof the NPAR agonist is added to the culture to provide more rapid growthor to maintain greater functionality of the chondrocytes than in theabsence of the agonist, i.e., a “stimulatory amount”. Typically, betweenabout 0.1 μg/ml and about 100 μg/ml of NPAR agonist is used.

Chondrocytes cultured in the presence of an NPAR agonists can also beused to treat cartilage damage by administering a therapeuticallyeffective amount of the chondrocytes to the site in need of treatment.With respect to chondrocytes, “therapeutically effective” also meanswhich results in greater cartilage growth or repair with the treatmentthan in its absence. The administration of chondrocytes to treatcartilage damage is described in U.S. Pat. No. 4,846,835, the entireteachings of which are incorporated herein by reference.

Thrombin peptide derivatives can be synthesized by solid phase peptidesynthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or byother suitable techniques including combinations of the foregoingmethods. The BOC and FMOC methods, which are established and widelyused, are described in Merrifield, J. Am. Chem. Soc. 88:2149 (1963);Meienhofer, Hormonal Proteins and Peptides, C. H. Li, Ed., AcademicPress, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E.Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp.3-285. Methods of solid phase peptide synthesis are described inMerrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G.Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis,5: 315 (1992)). The teachings of these six articles are incorporatedherein by reference in their entirety.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXEMPLIFICATION

Details of Experiments

Chondrocytes are the primary cell type found in cartilage. In cartilagethese cells are normally quiescent, or non-proliferative, and haverelatively low metabolic rates. Following injury to cartilage thesecells do not readily participate in the repair process. Due to theavascular nature of cartilage, these cells presumably would not seethrombin as an initiator of the repair process. The following examplesdemonstrate that chondrocytes have thrombin receptors and that compoundsthat activate NPAR stimulate chondrocyte proliferation and synthesis ofmatrix proteoglycans.

EXAMPLE 1 Thrombin Binding to Rat Chondrocytes

Primary cultures of rat articular chondrocytes were isolated andprepared for in vitro analysis using established methods (see Kuettner,K E., et.al., J. Cell Biology 93: 743-750, 1982). Briefly, cartilagepieces were dissected from the shoulder of rats and the pieces weredigested with trypsin for one hour and with collagenase for three hoursin tissue culture medium (DMEM) at 37 C. with stirring. The cells wereplated in flasks at high density (50,000 cells/cm sq.) and were culturein DMEM containing antibiotics an ascorbic acid at 37° C. in anatmosphere of 5% CO₂.

The specific binding of ¹²⁵I thrombin to chondrocytes was carried outusing established thrombin receptor binding assays as disclosed in U.S.Pat. No. 5,352,664 and Carney, D H and Cunningham, DD, Cell15:1341-1349, 1978. Briefly, highly purified human thrombin wasiodinated and added to cultures of chondrocytes with or withoutunlabeled thrombin to correct for nonspecific binding. By incubatingcells with different concentrations of labeled thrombin and measuringthe amount of thrombin bound to cells and the amount of free thrombin inthe medium it is possible to estimate the number of receptors per celland the affinity of thrombin for that binding site.

Scatchard analysis of the labeled thrombin binding from three separateexperiments suggest that rat chondrocytes express an average of 3000very high affinity binding sites (100 pM affinity) and 230,000 highaffinity sites (27 nM).

EXAMPLE 2A NPAR Agonist Stimulation of Bovine Chondrocyte Proliferation

Primary cultures of bovine chondrocytes were prepared using theprocedure described for rat chondrocytes in Example 1. The cultures weresubcultured into 24 well plastic dishes at a low density and placed in1% serum. Addition of the NPAR agonist TP508 to these cultures atconcentrations of 1.0 or 10 μg/ml by itself did not stimulate cellproliferation. In contrast, addition of these concentrations of TP508together with a small amount of thrombin co-mitogen, resulted in asmall, but significant (p<0.05) increase in cell number relative to thatseen in thrombin alone after three days in culture.

EXAMPLE 2B NPAR Agonist Stimulation of Bovine Chondrocyte ProteoglycanSynthesis

To determine the effect of NPAR agonists on proteoglycan synthesis,bovine chondrocytes were seeded into 96 well plates at a density of2×105 cells per well and cultured in DMEM with 10% fetal calf serum.After establishment of these multi-layer cultures, the medium wasreplaced daily with DMEM containing 1% serum with indicatedconcentrations of TP508 from 1 to 100 μg per ml (Table 1). After 6 daysin culture with daily changes of culture medium with or without TP508,³⁵S sulfate was added to the medium and incubation continued for anadditional 24 hours. As shown in Table 1, treatment with highconcentrations of TP508 (100 μg per ml) increased ³⁵S sulfateincorporation relative to untreated cells by more than 10-fold.

TABLE 1 Effect of the NPAR agonist TP508 on ³⁵S sulfate incorporation inbovine chondrocyte cultures. Mean CPM Treatment 1% Serum Std. Dev ofMean Control 4975 3552 TP508 (1 μg/ml) 4701 2692 TP508 (10 μg/ml) 69603265 TP508 (100 μg/ml) 81946 13783

EXAMPLE 3A NPAR Agonist Stimulation of Proliferation Synthesis inCultured Rat Articular Chondrocytes

Rat articular chondrocytes were isolated from slices of rat articularshoulder cartilage utilizing trypsin and collagenase digestions asdescribed in Example 1. Preparations of chondrocyte “3-dimensional”alginate bead cultures were established using established techniques asdescribed by Guo et. al., (Conn. Tiss. Res. 19:277-297, 1998). Followingremoval of cells from tissue culture flasks with trypsin, the cells weresuspended in an alginate gel (1.2% w/v) and slowly expressed through a22 gauge needle in a dropwise fashion into 102 mM CaCl₂. As the dropscontact the CaCl₂ there is a nearly instantaneous polymerization of thealginate to create a gel bead. The beads were then washed three times inDMEM culture medium and transferred to 35 mm dishes and maintained inculture at 37 C. in a 5% CO2 atmosphere by feeding with culture mediumevery two days.

The effect of NPAR agonist TP508 on chondrocyte cell proliferation afterthree days in 3-dimensional alginate culture was determined by removingbeads from 35 mm dishes, washing them with 0.9% saline, and dissolvingthe alginate beads by adding 1 ml of 55 mM sodium citrate, 0.15 M NaClat 37° C. for 10 minutes. Cell number was determined by diluting the 1ml of dissolved beads 1:10 with phosphate buffered saline (PBS) andcounting the cells with a Z-series Coulter Counter. As shown in Table 2,TP508 by itself stimulated proliferation of chondrocytes in 3dimensional culture.

TABLE 2 Effect of the NPAR agonist TP508 on Proliferation of RatChondrocytes in 3-D Bead Culture. Cells/bead Std. % Increase TreatmentAfter 3 days dev over Control Control 6238 688 TP508 30 nM 7463 167 19.7TP508 300 nM 8882 148 42.4 TP508 3 μM 8866 4 42.1 TP508 30 μM 7772 25824.6

EXAMPLE 3B NPAR Agonist Stimulation of Proteoglycan Synthesis inCultured Rat Articular Chondrocytes

To determine the effects of the NPAR agonist TP508 on proteoglycansynthesis, 3-dimensional alginate cultures were prepared as describedabove and assayed for incorporation of [³⁵S]-sulfate. Bead cultures wereexposed to indicated concentrations of TP508 as well as [³⁵S]-sulfate(20 μCi/ml) and with daily medium changes and were harvested on days 7for [³⁵S]-sulfate incorporation. At each time point 5-10 beads wereremoved, washed 3× with 0.9% saline, dissolved by adding 0.5 ml of 55 mMsodium citrate, 0.15 M NaCl at 37 C. for 10 minutes as described above,and counted in a liquid scintillation counter. [³⁵S]-sulfateincorporation was normalized in each sample for number of beads added.As shown in Table 3, TP508 treatment alone at a concentration of 300 nM(about 0.7 μg per ml), stimulated [³⁵S]-sulfate incorporation about 50%over controls. There was also a large stimulation by 30 μM TP508 (about70 μg per ml), however, there was a large relative standard deviation inmeasurements at this concentration.

TABLE 3 Effect of the NPAR agonist TP508 on [³⁵S]-sulfate incorporationinto proteoglycans. Std. % Increase Treatment CPM/bead dev over ControlControl 665 24 TP508 30 nM 829 87 24.7 TP508 300 nM 1008 29 51.6 TP508 3μM 827 9 24.1 TP508 30 μM 1153 519 73.3

EXAMPLE 4 Preparation of Polylactic Acid/Polyglycolic Acid CopolymerMicrospheres of TP508

A double emulsion technique was used to prepare microspheres ofpolylactic acid/polyglycolic acid copolymer (PLGA) containing TP508.Briefly, the matrix components were dissolved in methylene chloride andTP508 was dissolved in water. The two were gradually mixed togetherwhile vortexing to form a water-in-oil (W/O) emulsion. Polyvinyl alcohol(0.3% in water) was added to the emulsion with further vortexing to formthe second emulsion (O/W), thereby forming a double emulsion: an O/Wemulsion comprised of PLGA droplets, and within those droplets, a seconddisperse phase consisting of TP508 in water. Upon phase separation, thePLGA droplets formed discrete microspheres containing cavities holdingTP508. To cause phase separation of the microspheres, a 2% isopropylalcohol solution was added. The particles were collected bycentrifugation, and then lyophilized to remove residual moisture. Thecomposition of the matrix was varied to form microspheres with differentrelease kinetics (Table 4).

TABLE 4 Composition of different microsphere formulations Polymer % %polyethylene Formulation PLA:PGA M. Wt. TP508 glycol A 50:50 46,700 5 0B 50:50 7,200 5 0 C 50:50 46,700 5 5 D 50:50 46,700 5 0 E 75:25 120,0005 0

The mean diameter of the microspheres was measured in a Coulter counterand the drug entrapment efficiency was measured by spectrophotometricassay at 276 nm following dissolution of a weighed sample ofmicrospheres in methylene chloride and extraction of the released druginto water (Table 5).

TABLE 5 Formulation diameter and drug entrapment efficiency FormulationDiameter, μm TP508 Entrapment, % A 26.0 53.8 B 16.2 27.1 C 17.6 58.9 D23.9 42.6 E 25.8 36.2

To measure TP508 release from the different PLGA matrices, 20 mg ofmicrospheres were placed in 1.0 ml of PBS contained in 1.5 mlpolypropylene microcentrifuge tubes. Tubes were incubated at 37° C. andshaken at 60 rpm. At various times, the tubes were centrifuged and thesupernatant containing released TP508 was removed and frozen forsubsequent analysis. Fresh PBS was added to the microspheres andincubation was continued. TP508 in the supernatant was measured byabsorbance at 276 nm. For each formulation, quadruplicate releasedeterminations were performed. Formulations B and D showed no detectabledrug release during 28 days of incubation at 37° C. The remainingformulations all released detectable amounts of TP508, although in allcases the amount of drug released fell below detectable limits (<1 μg/mgmatrix/day) within 3-4 days. Formulations A and C showed the greatestrelease of TP508, releasing 60-80% of the entrapped drug over 3-4 days.Formulation C showed the fastest release kinetics and was chosen fortesting in the rabbit cartilage defect model described in Example 5.

EXAMPLE 5 The NPAR Agonist TP508 Stimulates Cartilage Growth in RabbitModels

Young, male New Zealand rabbits (2-3 kilograms) (n=15) were anesthetizedand given bilateral, medial longitudinal parapatellar arthrotomies. Theskin, subcutaneous tissue and joint capsule were incised, usingelectrocautery to minimize bleeding. The joint surface was exposed bylateral dislocation of the patella. A 3-mm diameter, 1-2-mm deepfull-thickness defect was made in the trochlear groove of the femurusing a surgical drill and pointed stainless steel drill bit. The aimwas to extend the defect into the subchondral plate without piercing thesubchondral bone.

The rabbits were divided into three groups. For each rabbit, both rightand left trochlear groove defects were filled with the same treatment.For this study, TP508 was formulated into PLGA controlled releasemicrospheres, prepared as described in Example 4 (Formulation C). Themicrospheres were mixed with sufficient Pluronic F68 gel (5% w/v) tobind the spheres together into a paste-like consistency that couldeasily be packed into the defect. The control group received PLGAmicrospheres without TP508 in both defects. The treated groups receivedmicrospheres containing either 10 or 50 mg of TP508/defect. One rabbitfrom each group was sacrificed at 4 weeks, 2 from each group weresacrificed at 6 weeks and the remaining animals were sacrificed at 9weeks. Samples were fixed and processed for histological analysis.

At the time of sacrifice, there appeared to be considerable fibrousgranulation tissue and no evidence of white cartilage-like material inthe control defects. In contrast, the defect had a nearly uniform,dense, white material filling in the defects from the 10 μg treatedgroup and 50 μg group. By 6 weeks post-surgery, the macroscopicdifferences between treated and control defects were not so pronounced.

Histology of the four week samples showed that indeed the controldefects were filled with what appeared to represent early granulationtissue including inflammatory and fibroblastic cells. In contrast, the10 and 50 microgram treated defects appeared to have a large number ofchondrocytes and early signs of cartilage formation. This effect wasseen more dramatically at week six. Controls had a small amount ofconnective tissue, yet little evidence of cartilage repair. In contrast,in both the 10 μg and 50 μg treated defects, there appeared to be goodintegration with hyaline cartilage forming at the top of the defect andextensive subchondral bone repair.

Nine-week TP508 treated defects exhibited a predominantly hyaline matrixwith evidence of significant aggrecan content as shown by positivesafranin-O staining. In most instances there was no difference inaggrecan content between the repair site and native tissue. Histologicalresults were quantitatively assessed using a grading system adapted byFreed, et al., J. Biomed. Materials Res. 28:891-899 (1944) from thescheme of O'Driscoll, et al., J. Bone Joint Surg. 126:1448-1452 (2000)with a maximum score of 25 for normal articular cartilage. ExperimentalTP508 treated defects scored mean averages that were significantlyhigher than control defects (Table 6).

TABLE 6 Histology Scoring For Articular Defect Study Milligrams of TP508Repair Score ± SE 0  9.4 ± 1.6 10 18.6 ± 1.4 50 19.8 ± 1.0

Peptide treated defects repaired with smooth articular surfaces and weretypically well bonded at the junction between repair and native tissue.The quality of control repair tissue was characterized as mostlyfibrocartilage with poor quality joint surfaces. Integration at thejunction between repair and native tissue was usually poor. Overall, thequality of cartilage repaired with TP508 was significantly enhanced overcontrol non-treated defects. This improved quality of repair tissueshould lead to more durable and functional restoration of jointbiomechanics and reduction in the incidence of osteoarthritis inpatients suffering from traumatic cartilage injuries.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of stimulating cartilage growth at an arthritic joint in asubject, said method comprising the step of administering to the site atherapeutically effective amount of the peptideAla-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 7).
 2. A method of stimulating cartilage growth in a subjectat a site being treated for cartilage loss, said method comprising thestep of administering to the site a therapeutically effective amount ofthe peptideAla-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 7).
 3. A method of stimulating cartilage growth in a subjectat a site being treated for cartilage loss due to traumatic injury, saidmethod comprising the step of administering to the site atherapeutically effective amount of the peptideAla-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 7).
 4. A method of stimulating cartilage growth or repair ata site in a subject in need of such growth or repair, said methodcomprising the step of administering to the site a therapeuticallyeffective amount of the peptideAla-Gly-Try-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO: 7).